Coagulation diagnostics are divided into global tests for examining the functionality of the blood coagulation cascade and individual tests for determining the activity of individual blood coagulation factors. Different test formats are known for both global tests and individual tests. Test formats are essentially divided into coagulation tests and chromogenic tests.
In a coagulation test, the patient sample to be examined, which usually consists of plasma, is mixed with a coagulation activator which starts the coagulation process. The coagulation process leads to the formation of a fibrin clot which can be measured with the aid of photometric methods. The rate of fibrin formation is a measure of the functionality of the blood coagulation cascade. To determine the activity of an individual coagulation factor in a coagulation test, the patient sample to be examined is additionally mixed with deficient plasma which supplies all components of the blood coagulation cascade except the blood coagulation factor to be tested.
In a chromogenic test, the patient sample to be examined, which usually consists of plasma, is mixed with a coagulation activator and with a substrate for a coagulation factor. Since most blood coagulation factors are serine endopeptidases, i.e., hydrolases which can cleave peptide bonds, use is predominantly made of peptide substrates which are cleaved highly specifically by the blood coagulation factor to be determined and which have a detectable signaling moiety. It may be preferred to use cleavable chromogenic or fluorogenic signalling moieties which are determined photometrically. Patent documents EP 34122 A1 and U.S. Pat. No. 4,508,644 describe a multiplicity of chromogenic peptide substrates and the use thereof in coagulation diagnostic tests, e.g., for determining the coagulation factors factor IIa (thrombin) and Xa. In document EP 78764 A1, a chromogenic method for determining the coagulation factor XIIa is described.
Particularly chromogenic tests can also be used to determine anticoagulants, which inhibit the activity of blood coagulation factors, in patient samples. For this purpose, the patient sample to be examined is mixed with an activated coagulation factor and with a substrate for this coagulation factor. The more anticoagulant that is present in the sample, the stronger is the inhibition of the activated coagulation factor and the less substrate that is cleaved.
These known methods are homogeneous methods. Homogeneous test methods have the advantage that the sample is mixed with the detection reagents to form a test mix and that the detection reaction is measured in this test mix without the need for additional separation steps, for example, for separating the analyte from the rest of the sample constituents. Homogeneous test methods, however, also have the disadvantage that substances intrinsic to the sample are present during the entire test method and, as a result, can affect the detection reaction or can interfere with the measurement of the detection reaction. Patient samples can, for example in individual cases, contain abnormally high concentrations of one or more intrinsic, i.e., endogenous substances, which can prove disruptive on exceeding a tolerable concentration in photometric detection methods and can lead to a systematic error. It is well known that problems are caused by hemolytic, icteric, and/or lipemic serum or plasma samples, so-called HIL samples which have abnormally high concentrations of hemoglobin, bilirubin, and/or triglyceride. Abnormally high concentrations of these interfering substances can be caused by a pathological state of the patient or else by inappropriate sample acquisition or storage.